Directed Evolution's Selective Use of Quantum Tunneling in Designed Enzymes─A Combined Theoretical and Experimental Study.

Natural enzymes are powerful catalysts, reducing the apparent activation energy for reactions and enabling chemistry to proceed as much as 10 times faster than the corresponding solution reaction. It has been suggested for some time that, in some cases, quantum tunneling can contribute to this rate enhancement by offering pathways through a barrier inaccessible to activated events. A central question of interest to both physical chemists and biochemists is the extent to which evolution introduces mechanisms below the barrier, or tunneling mechanisms. In view of the rapidly expanding chemistries for which artificial enzymes have been created, it is of interest to see how quantum tunneling has been used in these reactions. In this paper, we study the evolution of possible proton tunneling during C-H bond cleavage in enzymes that catalyze the Morita-Baylis-Hillman (MBH) reaction. The enzymes were generated by theoretical design, followed by laboratory evolution. We employ classical and centroid molecular dynamics approaches in path sampling computations to determine whether there is a quantum contribution to lowering the free energy of the proton transfer for various experimentally generated protein and substrate combinations. These data are compared to experiments reporting on the observed kinetic isotope effect (KIE) for the relevant reactions. Our results indicate the modest involvement of tunneling when laboratory evolution has resulted in a system with a higher classical free energy barrier to chemistry (that is, when optimization of processes other than chemistry results in a higher chemical barrier).